Thre overall goal of our laboratory is to elucidate the mechanisms controlling cell proliferation. This proposal concentrates on the Drosophila gene expanded (ex) which encodes a member of the protein 4.1 family of membrane-associated proteins. This gene is unusual in that its effect on cell proliferation is region-specific. Loss-of- function ex mutations cause an increase in the size of the wing, whereas general over-expression of the normal gene causes a decrease in size. Thus, appropriate levels of Ex expression are important for the local control of cell proliferation and final wing morphology. The predicted structure of Ex and the ex mutant phenotype suggest that Ex functions in a signaling pathway. This possibility is being tested by identifying genes and proteins that interact with Ex using both genetic and biochemical approaches. The yeast two-hybrid system has been used to screen Drosophila cDNA libraries for clones encoding Ex- binding proteins, and to test specific proteins that are already suspected to interact with Ex. Direct binding to Ex will be verified, and the genes encoding Ex-binding proteins will be identified and characterized by genetic and molecular methods. The domain in Ex responsible for its localization to the apical cell-membrane will be identified by expressing germline constructs encoding epitope-tagged domains of Ex, and determining their subcellular localization. The hypothesis that Ex acts as a linker molecule will be explored by examining the phenotypes induced by over-expression of these domains. The work will open up a new avenue of research into the mechanisms that control local differences in the rate and amount of cell proliferation in developing organs.